Development of a process for the production of the anticancer lead compound pleurotin by fermentation of Hohenbuehelia atrocaerulea.

Pleurotin is a naphthoquinone antibiotic originally isolated from Pleurotus griseus. Two pleurotin producing strains of Hohenbuehelia atrocaerulea have been identified, which, on solid substrate fermentation for 2 months yield 1-2 mg/l of the antibiotic. Described here is the lengthy developmental process which resulted in a production protocol being developed which reliably yields pleurotin from liquid fermentation at >300 mg/l. Critical to obtaining this increase in titer was inclusion in the media of an aqueous extract of alder wood.

Detecting potential teratogenic alkaloids from blue cohosh rhizomes using an in vitro rat embryo culture.

The novel alkaloid thalictroidine (1), as well as the known alkaloids taspine (2), magnoflorine (3), anagyrine (4), baptifoline (5), 5,6-dehydro-alpha-isolupanine (6), alpha-isolupanine (7), lupanine (8), N-methylcytisine (9), and sparteine (10), were identified from an extract of Caulophyllum thalictroides rhizomes. N-Methylcytisine exhibited teratogenic activity in the rat embryo culture (REC), an in vitro method to detect potential teratogens. The structure of 1 was elucidated using various spectroscopic methods, primarily by NMR techniques. Thalictroidine, anagyrine, and alpha-isolupanine were not teratogenic in the REC at tested concentrations. Taspine (2) showed high embryotoxicity, but no teratogenic activity, in the REC.

Production of brefeldin-A.

Fermentation conditions are described for the production of the antitumor antibiotic 7-(S)-brefeldin-A (brefeldin-A) in liquid culture by Eupenicillium brefeldianum, (B.Dodge) Stolk and Scott, ATCC 58665. An analytical hplc method was developed which allowed rapid quantitation of the compound during fermentation. A kilogram of brefeldin-A was isolated from a fermentation at the 6800-liter scale.

Micellar electrokinetic chromatography of the anti-human immunodeficiency virus (HIV) agent michellamine B with absorption and laser-induced fluorescence detection.

Micellar electrokinetic chromatography (MEKC) was applied to the separation of the anti-HIV agents, michellamines A and B, and two other structurally related monomers found in the extract of the Ancistrocladus plants. Using buffers containing either 10 mM sodium phosphate (pH 7.0), 50 mM sodium deoxycholate and 10-20% acetonitrile or 5 mM sodium phosphate (pH 7.0), 20 mM sodium dodecyl sulfate and 25% acetonitrile allowed baseline separations of the four components in the mixture in less than 10 min. The MEKC methods gave sharper peaks and better resolution compared to high-performance liquid chromatography. For MEKC separation of the plant extracts, UV absorption detection provided adequate sensitivity; however, higher sensitivity could be achieved with UV laser-induced fluorescence detection (LIF). Using the sodium dodecyl sulfate-containing buffer and LIF, the limit of detection for michellamine B was approximately 2 ng/mL. The sensitivity was degraded approximately 100-fold when using the deoxycholate buffer because of high background fluorescence. Preliminary results show that MEKC with LIF is feasible for the sensitive detection of michellamine B in serum.

Ethnobotany and drug discovery: the experience of the US National Cancer Institute.

Between 1960 and 1981 the National Cancer Institute (NCI) screened 114,000 extracts of 35,000 plants, mainly collected in temperate regions. Of the three clinically active anticancer drugs so far discovered in that programme, none was isolated from a plant collected on an ethnobotanical basis, though various Taxus species, which are the source of taxol, are reported to have been used medicinally. Since 1986, the NCI has focused its collections in tropical and subtropical regions worldwide; collections cover a broad taxonomic range, though priority is given to medicinal plants when relevant information is available. As of August 1993, 21,881 extracts derived from over 10,500 samples had been tested in a screen for activity against the human immunodeficiency virus (HIV); 2320 of these extracts were of medicinal plant origin. Approximately 18% of both the total number of extracts and the medicinal plant-derived extracts showed significant anti-HIV activity; in each instance about 90% of the active extracts were aqueous. The activity of the aqueous extracts has been attributed mainly to the presence of polysaccharides or tannins. Four plant-derived compounds are in preclinical development at the NCI; only one of the four sources plants, obtained from a noncontract source, was collected on an ethnobotanical basis. At this stage the results indicate that the current NCI collection policy offers the best chances for the discovery and development of agents for the treatment of AIDS (acquired immune deficiency syndrome) and cancer.

Dereplication of phorbol bioactives: Lyngbya majuscula and Croton cuneatus.

Lyngbya majuscula and Croton cuneatus were used as prototypes for the dereplication of phorbol ester receptor binding activity using a combination of hplc-uv and online phorbol dibutyrate (PDBu) receptor binding and batch fractionation over either Si gel or diolbonded Si gel. Debromoaplysiatoxin was responsible for the bioactivity of Lyngbya, whereas a complex of potent phorbol esters was detected in C. cuneatus.

Annonacin, a novel, biologically active polyketide from Annona densicoma.

A new linear polyketide, Annonacin (I), has been isolated from active extracts of the stembark of Annona densicoma Mart. Annonacin (I) is highly cytotoxic and is active in an assay designed to detect antimitotic agents. The structure of (I) was determined by analysis of spectroscopic data.

New xanthones from Psorospermum febrifugum.

The isolation and structural determination of two new xanthone derivatives, 1,2,4-trimethoxy-3,8-dihydroxyxanthone, 1,2,4-trimethoxy-3-hydroxyxanthone and a known 2-methoxy-3-hydroxyxanthone from Psorospermum febrifugum var. ferrugineum are reported. The structures were elucidated by extensive analysis of 1H and 13C nmr, ms, and chemical correlations.

Antineoplastic agents from higher plants: application of tandem mass spectrometry to xanthones from Psorospermum febrifugum.

Psorospermum febrifugum was examined using tandem ms for the presence of 3',4'-deoxypsorospermin-4'-chloro-3'-ol and psorospermin. Collision-induced dissociation at both high and low energy is used to bring about fragmentation. Daughter spectra were interpreted to reveal characteristic fragmentations for both compounds. Examination of the authentic compounds, various extracts of the plant root, and the plant root material itself established that psorospermin occurs naturally in the plant. The data also suggest the presence of the chlorohydrin in the root, as well as establishing its presence in the root extracts. The detection of neutral compounds in complex mixtures by tandem ms is facilitated if comparisons can be made between spectra resulting from different methods of ionization and excitation.

Immunological and biophysical separation of dengue-2 antigens.

Antigenic compositions of slowly sedimenting dengue-2 hemagglutinin (SHA) and soluble complement-fixing antigen (SCF) were compared with the virion (rapidly sedimenting hemagglutinin, RHA) by radioimmune precipitation (RIP), RIP inhibition, kinetic neutralization, and neutralization blocking tests with the use of hyperimmune mouse ascitic fluids. RHA and SHA were unable to inhibit completely the RIP of each other by anti-RHA, and neutralization by anti-RHA was not blocked by SHA. This indicated that SHA is serologically related, but not identical, to RHA. SHA differed from RHA in that SHA lacked the "core" polypeptide but contained the two envelope polypeptides. In addition, SHA contained a polypeptide with a molecular weight of 16,500 daltons and a suggestion of several other proteins. These data, when considered with other evidence, suggest that SHA is a special form of "incomplete virus." SCF was unable to inhibit the RIP of SHA or RHA or to block neutralizing antibodies. Further, anti-SCF did not neutralize RHA or precipitate significant levels of SHA or RHA. Polyacrylamide gel electrophoresis separated SCF from structural polypeptides by molecular size. This evidence suggests that SCF is a nonstructural antigen.